Enzyme kinetics

Question 1

Describe the Michaleis-Menton equation

Answer 1

used for large amount of enzymes, also called Michaleis enzymes to determine the dependence of the enzymatic activity on the substrate concentration

S + E -> ES -> P + E

S = substrae, E = enzyme, ES = enzyme-substrate complex, P = product

V = velocity of the enzyme reaction

[S] = concentration of the substrate (molar concentration)

VMax = maximum velocity, corresponding to the saturated substrate concentration

KM (Michaleis constant) = is the [S] at which the reaction reaches half its maximum velocity

KM is the measure of the affinity of enzyme for S

The lower the Km, the higher the affinity of the enzyme for the substrate and vice versa (inverse proportional)

Question 2

Linewaewer-Burk equation

Answer 2

used to determine the kinetic parameters of the enzymes => the linearized form of the Michaleis-Menton equation (Linewaver-Burk relation)

used to determine K_M and V_MAX

Question 3

Differences in both equations

Answer 3

Question 4

Pseudocholinesterase

(Cholinesterase): reference range

Answer 4

serum pseudocholinesterase activity is 4-10 U/ml

Question 5

Medical importance of pseudocholinesterase

Answer 5

is an imporatant marker of liver function

Serum cholinesterase activity decreases in:

• hepatic failure: hepatic cirrhosis, liver insufficiency
• intoxication with organophosphorous compounds

Question 6

Important enzymes and frequently used in diagnosis

Answer 6

Transaminases (GOT, GPT)

Amylase

Acid and alkaline phosphatase

Gamma-glutamyl transferase (GGT)

Question 7

Transaminases (GOT, GPT)

Answer 7

Reference range:

• Serum GOT: < 50 U/L
• Serum GPT: < 40 U/L

“OP”

medical importance:

• The activity of serum GOT increases in: acute hepatitis, toxic necrosis of the liver, myocardial infarction
• The activity of serum GPT increases in: acute hepatitits, toxic necrosis of the liver

Question 8

Amylase

Answer 8

reference range:

Serum amylse: 60-300 U/L

medical importance:

serum amylase activity increases in case of

• acute pancreatitis
• parotitis

“PP”

Question 9

Acid and alkaline phosphatase

Answer 9

reference range:

• Serum acid phosphatase: < 11 U/L
• Serum alkaline phosphatase: 20-48 U/L

medical importance:

• the activity of the serum acid phosphate increases in prostata cancer
• the activity of the serum alkaline phosphatase is increased in case of hepato-biliary obstruction (stones, tumors of bile canaliculi) and in bone diseases (the main sources alkaline phosphatase are the liver and bone)

Question 10

Gamma glutamyl transferase (GGT)

Answer 10

reference range:

Serum GGT: 8-50 U/L (male), 8-40 U/L (female)

medical importance:

Serum GGT activity increases in:

• hepatobilary disorder (bilary tract obstruction)
• alcoholism (GGT is sensitive to alcohol)
• drugs administration: phenobarbital, warfarin, phenytoin (they are enzyme inducing drugs)
• other conditions: acute pancreatitis, diabetes mellitus

Question 11

Acetylcholinesterase (true cholinesterase)

Answer 11

group of enzymes that belong to the class of hydrolases, a subclass of esterases which hydrolyze the esters of cholins

uses acetylcholine as substrate

acetylcholinesterase (AChE) actis on a specific substrate => acetylcholine is hydrolyzed (in the presensece of AChe) to choline and acetic acid

Question 12

Role of acetylcholine

Answer 12

is an important neurotransmitter, an abnormal hydrolisis of acetylcholine leads to an increased amount of this substrate (acetylcholine) =>

consequence is a block in neuromuscular activity

Question 13

Cholinesterase (nonspecific cholinesterase or pseudocholinesterase)

Answer 13

group of enzymes taht belong to the class of hydrolases, a subclass of esterases which hydrolyze the esters of cholins

uses different esters of cholin as a substrate

cholinesterase has a greater affinity for butyryl esters

cholinesterase hydrolyzed butyrylcholine to choline and butyric acid

Question 14

Pseudeocholinesterase

(Cholinesterase)

Answer 14

role: is an enzyme present in plasma and is synthesized by the liver

= important marker of liver function

Question 15

Principle of serum cholinesterase activity determination:

Answer 15

substrate: butyrylthiocholine
1. butyrylthiocholine is hydrolyzed in the

present of pseudocholinersterase (PChE) to thiocholine and butyric acid

2. The released thiocholine reacts DTNB to form yellow compound 5-thio-2-nitrobenzoic acid with maximum absoprtion at 412 nm

Question 16

Choose the correct answer:

a. The normal value of serum cholinesterase in is 20-48 U/ml;
b. The substrate used for serum cholinesterase activity determination is butyrylthiocholine

c. The activity of serum alkaline phosphate is decreased in hepato-biliary obstruction
d. Acetylcholinesterase use different esters of choline as substrate
e. The normal value of serum acid phosphate is >11 U/L

Answer 16

b. The substrate used for serum cholinesterase activity determination is butyrylthiocholine